Current single use (SU) small and large scale cell culture bioreactors used for development work in pilot plants or for full scale GMP production are typically designed with a vertical vessel that holds a disposable bag fully instrumented and with necessary gas flows and sterile inlet and outlet filters. In addition, many SU cell culture bioreactor mixer designs are limited in the power they are able to deliver into the culture fluid. The industry has been seeking to provide a single use microbial fermentor by leveraging what has been learned from the single use cell culture bioreactors on the market. Some single use vertical microbial fermentors have been introduced with a single drive shaft having multiple impellers. Single vertical shaft designs also utilize complicated internal shaft support bearings to stabilize the rotor. Magnetic drives that avoid dual mechanical shaft seals through the pre-sterilized bag are preferred. However the nature of the magnetic coupling that occurs limits the mixing power delivered to the fluid as compared to an equivalent stainless system using direct shaft drives having dual mechanical shaft seals.
Some current vertical single use fermentor designs have mixing limitations with respect to impeller tip speeds and shaft rotational speed and stability, particularly when the available impeller limitations might result in long mixing times, poor oxygen mass transfer, or restrictions on minimum operating level in the bag. Single use cell culture bioreactor systems have gained great success in mammalian cell applications (as an example culturing chinese hamster ovary (CHO) cells) to produce monoclonal antibodies (MaB's) where low power per unit volume and long mixing times are tolerable due to the slow metabolism of the cells. Cell culture bioreactors typically have batch runs of 21 to 30 days with relatively gentle mixing with low gas flows even during the maximum cell density stage where product formation occurs.
Vertical single use bags are being developed for microbial fermentation applications to varying degrees of success however there remains the challenge of delivering high power per unit volume and high oxygen transfer rates, particularly for the magnetic drive solutions. In addition these vertical form factor vessel systems at large scale (>1000 liters working volume) require significant accommodations for operator access to the top of the bag for installation and removal of the bag, the inlet and exhaust filters, and possible other components such as sensors and exhaust condensers at the top bag gas outlet. In very large systems at 1500 liter scale or greater, some facility consideration must be made for high ceiling stub-ups (greater than 9 ft.), mezzanines or permanent platforms to permit safe operator access.
There is a market need for a scalable single use microbial fermentor solution that is capable of delivering the necessary power per unit volume input, sparge air flows of 1 vessel volume per minute (VVM) or higher, optional sparge oxygen flow of 1 VVM, a pressurized environment and effective mixing times with attendant aggressive agitation to support the high oxygen mass transfer rate needed for microbial growth kinetics and metabolism of pharmaceutical recombinant therapeutic proteins, vaccines and other fermented products. In addition to the vertical single use cell culture bioreactors currently in use, other types of bioreactors such as rocker plate, rocker plus translation motion, orbital shaker, air lift/air wheel, packed bed, or other novel designs also have the inability to achieve high power per unit volume input to the liquid thereby making them ineffective for efficient fast microbial growth while they are quite satisfactory for slow growing mammalian cell cultures.
Current polymer bioreactor and fermentor bag holder designs with an open top container limit operating pressures to about 0.5 psig to prevent bag bursting that could cause injury to personnel and/or release of valuable and/or cytotoxic material to the environment. This low operating pressure, while not of consequence in cell culture bioreactors, does restrict oxygen mass transfer capability in microbial fermentations. Equivalent stainless steel fermentor systems can have relatively high operating pressures of typically 10 to 15 psig that contribute to driving force and high oxygen mass transfer required at relatively high cell densities thereby aiding metabolism.
Current vertical tank bag holders are conducive to single bottom or top mixer drives often with magnetic coupling to avoid breaching the bag boundary with a mechanical seal. The magnetic flux coupling between the drive magnets and the driven magnets limits torque transmission which in turn limits top speed, impeller tip speed, oxygen mass transfer, and number of impellers permitted on a given shaft. Typically as working volume increases to 500 liters and beyond, multiple impellers are needed to avoid stratification and these extended length shafts do not operate well at high speeds with a bottom or top magnetically coupled drive. In the case of top drives, multiple impellers are possible but vibration and critical speed issues due to longer shafts as well as power per unit volume input and top speed limitations makes them unsuitable for high cell density microbial fermentations.
Current large scale SU vertical bioreactors (1000 L and greater) are not conducive to use in modular clean room facilities where ceiling height limitations (typically 9 ft.) make scale-up more difficult. Vertical bag holders can easily exceed the 9 foot ceiling limitation of a typical modular building thus making special ceiling “top hats” or “stub-ups” necessary. This complicates the design and construction of modular facilities. Further, large scale, 1000 liter working volume and larger vertical SU bioreactor or fermentor designs are not portable or modular thus tending to remain fixed in place once the bioreactor suite is installed.
Current market drivers for single use technology in biopharmaceutical manufacture are: improved speed of production thus faster time to market, flexibility of equipment to easily adapt to process changes, avoidance of cleaning and steam sterilization, as well as a reduction in capital cost. Classic stainless steel multi-use microbial fermentors and mammalian cell culture bioreactors have been used in the Pharmaceutical and Biotechnology industries for over 40 years. Many drug production companies have FDA validated processes thus their existing infrastructure and stainless steel equipment remain their dominant and/or preferred design.
The market for SU cell culture bioreactors at lab, process development and even large production scale has and will continue to grow as these designs prove to meet the demands of the industry. Mammalian cells and other eucaryotic cells continue to dominate the expression systems needed to produce large quantities of monoclonal antibodies and biotherapeutic proteins.
By contrast current microbial fermentors in the single use market have limitations inherent in their design ranging from use of unpressurized or very low pressure disposable bags and magnetic drives that do not achieve equivalent mixing and mass transfer of oxygen as their stainless steel counterparts can deliver.
Some vertical form factor fermentors are on the market up to 300 liters working volume that claim adequate mixing and oxygen mass transfer to achieve successful microbial growth and product formation. Newly released 1000 L single use fermentors are being introduced however there is very little in the way of actual production use to date. Microbial cells can be nurtured and grown to produce relatively high density cell mass. This cell mass then can produce high titers of metabolites (products of cell metabolism) or produce proteins when the cells have been genetically engineered to produce large quantities of a specific recombinant protein. Large product quantity requires the use of large scalable fermentors in the 1000 liter to 3000 liter working volume range. Current single use microbial fermentors cannot scale to this size leaving a void in the production capacity of these systems.
The products made in this type of equipment range from perfume, fuel, and amino acids to a wide array of vaccines, therapeutic recombinant proteins, monoclonal antibodies, and various sophisticated fusion proteins or other polypeptides.
The fermentor systems whether they are stainless steel or single use require instruments, piping components, manual and automatic valves, gas flow control, agitators for mixing and oxygen mass transfer, temperature control modules, pressure control, liquid addition and harvest systems, overpressure safety systems, and a range of digital controllers properly configured to monitor and control the microbial process successfully. Successful operation is defined as controlled metabolism of the organism during cell doubling followed by proper production or expression of the product of interest. The current single use systems needed to efficiently grow and metabolize mammalian cells have copied from stainless steel vertical bioreactor vessels having a single agitator with one or several impellers coupled to a single shaft drive system.
Attempts are being made to create single use microbial fermentors using previously mentioned rocker plates, orbiting shakers, airlift wheels, paddle mixing and other novel designs, none of which are capable of achieving the necessary scalability in terms of input power per unit volume. The current vertical fermentor single use systems have limited power per unit volume capability upon scale-up result in inadequate mixing and oxygen mass transfer particularly since they are non-pressurized systems. In addition larger size single use systems result in very tall vessels that require special considerations when fitting into clean rooms, modular construction buildings or other portable type production facilities. Use of ladders, platforms, or other super-structures complicate access to the fermentor and may add to operator turn-around time between batches.
In addition the current vertical form factor for single use fermentors prohibits the possibility of portability that is a prerequisite for rapid deployment when a therapeutic recombinant protein or vaccine is needed in a fast time frame as an example, medical counter measures (MCM) in the event of a biological toxin release.
Further, culture of microorganisms in single use bags creates a conundrum since said microorganisms are grouped in various biosafety levels for large scale production (greater than 10 liter batches). The National Institutes of Health (NIH) specifies physical containment levels and defines Biosafety Levels for Large Scale in their “Guidelines for Research Involving Recombinant DNA Molecules” (NIH Guidelines)—Appendix K—“Physical Containment for Large Scale Uses of Organisms Containing Recombinant DNA Molecules”. April 2002. These categories range from safest to most hazardous in the following categories, Good Large Scale Practice (GLSP), BL1-LS, BL2-LS, and BL3-LS. These categories closely match the Center for Disease Control (CDC) categories but the CDC also includes a fourth category, BSL4 however these types of cultures are not recommended for use in any type of bag or single use container due to their inherent hazard to human exposure.
Current single use disposable fermentors have not addressed any type of secondary containment in situations where BL2-LS and/or BL3-LS organisms may be needed for production purposes.